Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).     

N-(Carboxyacyl)chitosans

Hemicelluloses were isolated from pineapple-leaf fibers under different conditions. Study of the properties of these hemicelluloses gave direct evidence of some ester linkages between the hemicellulose and the lignin in this fiber. An aldobiouronic acid was isolated from this fiber hemicellulose, and characterized as 2-O-(4-O-methyl-α-d-glucopyranosyluronic acid)-d-xylose. This indicates that the general nature of the hemicellulose is similar to those of jute and other fiber hemicelluloses.     

Characterisation of Authentic Lignin Biorefinery Samples by Fourier Transform Infrared Spectroscopy and Determination of the Chemical Formula for Lignin

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C₉H_10.2O_3.4 with slight variations depending on the biomass feedstock and processing conditions (between C₉H_9.5O_2.8 and C₉H_11.1O_3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm^?1 relative to the strong band at ~1120 cm^?1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.     

Enhanced enzymatic hydrolysis and structural features of corn stover by FeCl3 pretreatment

Corn stover was pretreated with FeCl₃ to remove almost all of the hemicellulose present and then hydrolyzed with cellulase and β-glucosidase to produce glucose. Enzymatic hydrolysis of corn stover that had been pretreated with FeCl₃ at 160 °C for 20 min resulted in an optimum yield of 98.0%. This yield was significantly higher than that of untreated corn stover (22.8%). FeCl₃ pretreatment apparently damaged the surface of corn stover and significantly increased the enzymatic digestibility, as evidenced by SEM and XRD analysis data. FTIR analysis indicated that FeCl₃ pretreatment could disrupt almost all the ether linkages and some ester linkages between lignin and carbohydrates but had no effect on delignification. The FeCl₃ pretreatment technique, as a novel pretreatment method, enhances enzymatic hydrolysis of lignocellulosic biomass by destructing chemical composition and altering structural features.     

Biodegradation of lignin-carbohydrate complexes

Covalent lignin-carbohydrate (LC) linkages exist in lignocellulose from wood and groups herbaceous plants. In wood, they consist of ester and ether linkages through sugar hydroxyl to the -carbanol of phenylpropane subunits in lignin. In grasses, ferulic and p-coumaric acids are esterified to hemicelluloses and lignin, respectively. Hemicelluloses also contain substitutents and side groups that restrict enzymatic attack. Watersoluble lignin-carbohydrate complexes (LCCs) often precipitate during digestion with polysaccharidases, and the residual sugars are more diverse than the bulk hemicellulose. A number of microbial esterases and hemicellulose polysaccharidases including acetyl xylan esterase, ferulic acid esterase, and p-coumaric esterase attack hemicellulose side chains. Accessory hemicellulases include -l-arabinofuranosidase and -methyl-glucuranosidase. Both of these side chains are involved in LC bonds. -Glucosidase will attach sugar residues to lignin degradation products and when carbohydrate is attached to lignin, lignin peroxidase will depolymerize the lignin more readily.Abbreviations APPL acid precipitable polymeric lignin - CBQase cellobioquinone oxidoreductase - LC lignincarbohydrate - LCC(s) lignin-carbohydrate complex - DHP Dehydrogenative polymerisate - DMSO dimethylsulfoxide - DP degree of polymerisation - MWEL milled wood enzyme lignin - MWL milled wood lignin (not digested with carbohydrases)     

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.     

阿魏酸酯酶和纤维素酶在水解汽爆稻草中的协同作用

利用阿魏酸酯酶, 水解天然木质纤维素原料中半纤维素与木质素之间的阿魏酸酯键, 从破坏两者共价键连接的角度, 探索阿魏酸酯酶促进纤维素酶水解汽爆稻草中纤维素的可行性。结果显示, 当阿魏酸酯酶加入量为240 mu/g底物、水解72 h时, 汽爆稻草纤维素的酶解率、不溶性底物失重率较不加阿魏酸酯酶分别增加了32.00%、32.77%; 阿魏酸酯酶(300 mu/g底物)作用120 min后, 纤维素酶对汽爆稻草纤维素的酶解率、不溶性底物失重率分别增加了29.85%、32.48%。通过比较不同酶法处理后的汽爆稻草的可及度和红外光谱图发现, 阿魏酸酯酶能有效地水解原料中的酯键, 提高原料可及度50%以上。由此表明, 阿魏酸酯酶和纤维素酶之间存在较大的协同作用, 添加阿魏酸酯酶能够提高纤维素酶对天然木质纤维素的酶解效率。     

Litter decay rates are determined by lignin chemistry

Litter decay rates are often correlated with the initial lignin:N or lignin:cellulose content of litter, suggesting that interactions between lignin and more labile compounds are important controls over litter decomposition. The chemical composition of lignin may influence these interactions, if lignin physically or chemically protects labile components from microbial attack. We tested the effect of lignin chemical composition on litter decay in the field during a year-long litterbag study using the model system Arabidopsis thaliana. Three Arabidopsis plant types were used, including one with high amounts of guaiacyl-type lignin, one with high aldehyde- and p-hydroxyphenyl-type lignin, and a wild type control with high syringyl-type lignin. The high aldehyde litter lost significantly more mass than the other plant types, due to greater losses of cellulose, hemicellulose, and N. Aldehyde-rich lignins and p-hydroxyphenyl-type lignins have low levels of cross-linking between lignins and polysaccharides, supporting the hypothesis that chemical protection of labile polysaccharides and N is a mechanism by which lignin controls total litter decay rates. 2D NMR of litters showed that lignin losses were associated with the ratio of guaiacyl-to-p-hydroxyphenyl units in lignin, because these units polymerize to form different amounts of labile- and recalcitrant-linkages within the lignin polymer. Different controls over lignin decay and polysaccharide and N decay may explain why lignin:N and lignin:cellulose ratios can be better predictors of decay rates than lignin content alone.     

Aromatic ring cleavage of β-O-4 lignin model dimers without prior demeth(ox)ylation by lignin peroxidase

Methyl oxalate of arylglycerol was formed as an aromatic ring cleavage product in degradation of arylglycerol-β-aryl ether (β-O-4) type lignin substructure model dimers by extracellular lignin peroxidase of Phanerochaete chrysosporium. The enzymatic cleavage of arylglycerol-β-(o-[²H₃]methoxyphenyl) ether indicated that the methyl group of the methyl ester was derived from the methoxy group of the β-O-4 model dimer. It is thus concluded that demeth(ox)ylation was not essential for the enzymatic aromatic ring cleavage of the methoxylated aromatic substrates, β-O-4 lignin substructure models.     

Isolation and Structural Characterization of Lignin Polymer from Dendrocalamus sinicus

Dendrocalamus sinicus, which is the largest bamboo species in the world, has broad prospects in the fields of bioenergy and biorefinery application. In this study, dewaxed D. sinicus samples were sequentially treated with 80 % ethanol containing 0.025 M HCl, 80 % ethanol containing 0.5 % NaOH, and aqueous alkaline solutions (containing 2.0, 5.0, and 8.0 % NaOH, respectively) at 75 °C for 4 h, in which 9.63, 8.71, 21.83, 21.09, and 13.09 % of the original lignin were isolated, respectively. The lignin fractions obtained were comparatively characterized by chemical composition, molecular weights, and structural features by wet chemical and instrumental analysis methods. It was found that the bamboo lignin fractions isolated by ethanol had lower weight-average molecular weights (1,360–1,380 g?mol^?1) and contained much higher amounts of associated hemicelluloses, while the lignin fractions isolated by aqueous alkaline solutions had higher weight-average molecular weights (5,300–6,040 g?mol^?1) and contained lower amounts of associated hemicelluloses. Spectroscopy analyses indicated that the bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. A small percentage of the lignin side-chain was found to be acetylated at the γ-carbon, predominantly at syringyl units. The major interunit linkages present in the bamboo lignin obtained were β-O-4′ aryl ether linkages, together with lower amounts of β-β′, β-5′, and β-1′ linkages.     

Search for optimum conditions of wheat straw hemicelluloses cold alkaline extraction process

A method for the selective extraction of hemicellulose from wheat straw involving cold alkaline extraction and subsequent separation by precipitation with ethanol is proposed. Wheat straw affords selective separation of the hemicellulose fraction from the cellulose and lignin fractions with the proposed method. The hemicellulose yield was optimized by using a 2n factor design to examine the influence of temperatures (temperature was designed between 20 and 40 °C), operation times (operation time was designed between 30 and 60 min) and alkali concentrations (alkali concentration was designed between 80 and 120 g L⁻¹). These conditions allowed 56.1% of all hemicellulose initially present in the raw material, and 59.1% of the lignin, to be extracted. Subsequent separation of hemicellulose in the liquid phase from the cold alkaline extraction by precipitation with ethanol provided a fraction containing 39.4% of all hemicellulose (45.2% hemicellulose in extract/total extract) and only 12% of all lignin in the raw material.     

Inhibition of trehalose breakdown increases new carbon partitioning into cellulosic biomass in Nicotiana tabacum

Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive ¹¹CO₂, and the partitioning of isotope was traced into [¹¹C]cellulose and [¹¹C]hemicellulose, as well as into [¹¹C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4 d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein.     

Compositional Characterization and Pyrolysis of Loblolly Pine and Douglas-fir Bark

Two potential biofuel resources, Douglas-fir and Loblolly pine bark, were subjected to extensive chemical and compositional analysis. The barks were initially extracted with dichloromethane, and the resulting extracted compounds were characterized by gas chromatography coupled with mass spectrometric analysis. Characterization of the major bark biocomponents indicated that Douglas-fir and Loblolly pine bark contained 22.5 and 13.2 % tannins, 44.2 and 43.5 % lignin, 16.5 and 23.1 % cellulose, and 7.6 and 14.1 % hemicellulose, respectively. Of particular interest is the high content of tannins and lignin, which make these barks excellent potential precursors for bio-oils and/or other value-added chemicals. ¹³C nuclear magnetic resonance (NMR) was used to characterize the chemical structure of the lignin and tannins. These samples were also analyzed by ³¹P NMR after phosphitylation of the hydroxyl groups in lignin and tannins. The NMR spectral data indicated that the lignin in both barks contained p-hydroxyphenyl (h) and guaiacyl (g) of lignin monomers with an h/g ratio of 10:90 and 22:78 for Douglas-fir and Loblolly pine bark, respectively. Gel permeation chromatography was used to analyze the molecular weight distributions of extracted tannins, isolated cellulose, and ball-milled lignin. The pyrolysis of Douglas-fir and pine bark at 500°C in a tubular reactor generated 48.2 and 45.2 % of total oil, of which the light oil contents are 14.1 and 20.7 % and heavy oil are 34.1 and 24.4 %. Similarly, fast pyrolysis at 375°C yielded 56.1 and 49.8 % of total oil for Douglas-fir and pine bark, respectively.     

Comparative ligninolytic and polysaccharolytic potentials of an alkaliphilic basidiomycete on native ligninocellulose

A comparison of the ligninolytic, cellulolytic and hemicellulolytic abilities of an alkaliphilic white-rot fungus. Coprinus fimetarius, on wheat straw under varying conditions of solid-substrate fermentation is presented. The extent of fractional degradation (percentage of the original dry weight of the fraction) of straw under an optimized set of cultural conditions (pH 9·0, moisture 65%, temperature 37°C, period 21 days) was in the following order: lignin (45%), cellulose (42%), hemicellulose (27%). Urea nitrogen favoured the degradation of lignin as well as cellulose and hemicellulose up to a certain level (1·5% sterile urea or 3% unsterile urea on a dry weight basis) beyond which the degradation of lignin was relatively more adversely affected than cellulose. The addition of phosphorus and sulphur was found essential for selective lignin removal. Increasing the C:N ratio by addition of free carbohydrates resulted in an overall decrease in the degradation wherein cellulose utilization was the most affected event. The pre-treatment (physical or chemical) of the substrate caused a general increase in biodegradation of lignin, cellulose and hemicellulose. The degrading activity of the fungus declined with the scaling-up of the fermentation particularly under non-sterile conditions.     

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin,glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.     

Characterisation of the initial degradation stage of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) sapwood after attack by brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

In our study, early period degradation (10 days) of Scots pine (Pinus sylvestris L.) sapwood by the brown-rot fungus Coniophora puteana (Schum.: Fr.) Karst. (BAM Ebw.15) was followed at the wood chemical composition and ultrastructurelevel, and highlighted the generation of reactive oxygen species (ROS). An advanced decay period of 50 days was chosen for comparison of the degradation dynamics. Scanning UV microspectrophotometry (UMSP) analyses of lignin distribution in wood cells revealed that the linkages of lignin and polysaccharides were already disrupted in the early period of fungal attack. An increase in the lignin absorption A₂₈₀ value from 0.24 (control) to 0.44 in decayed wood was attributed to its oxidative modification which has been proposed to be generated by Fenton reaction derived ROS. The wood weight loss in the initial degradation period was 2%, whilst cellulose and lignin content decreased by 6.7% and 1%, respectively. Lignin methoxyl (–OCH₃) content decreased from 15.1% (control) to 14.2% in decayed wood. Diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy corroborated the moderate loss in the hemicellulose and lignin degradation accompanying degradation. Electron paramagnetic resonance spectra and spin trapping confirmed the generation of ROS, such as hydroxyl radicals (HO^∙), in the early wood degradation period. Our results showed that irreversible changes in wood structure started immediately after wood colonisation by fungal hyphae and the results generated here will assist in the understanding of the biochemical mechanisms of wood biodegradation by brown-rot fungi with the ultimate aim of developing novel wood protection methods.     

Unveiling the Structural Heterogeneity of Bamboo Lignin by In Situ HSQC NMR Technique

One of the primary challenges for efficient utilization of lignocellulosic biomass is to clarify the complicated structure of lignin. In this study, in situ heteronuclear single quantum coherence nuclear magnetic resonance (NMR) characterization of the structural heterogeneity of lignin polymers during successively treated bamboo was emphatically performed without componential separation. Specially, the NMR spectra were successfully obtained by dissolving the acetylated and non-acetylated bamboo samples in appropriate deuterated solvent (CDCl₃ and DMSO-d ₆). The heterogeneous lignin polymers in bamboo samples were demonstrated to be HGS-type and partially acylated at the ??-carbon of the side chain by p-coumarate and acetate groups. The major lignin linkages (?¨CO?C4, ?¨C??, and ?¨C5, etc.) and various lignin?Ccarbohydrate complex linkages (benzyl ether and phenyl glycoside linkages) can be assigned, and the frequencies of the major lignin linkages were quantitatively obtained. In particular, the residual enzyme lignin (REL) contained a higher amount of syringyl units and less condensed units as compared to other samples. Inspiringly, the method gives us a vision to track the structural changes of plant cell wall (e.g., lignin polymers) during the different pretreatments.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

A note on utilization of bagasse for the production of proteinaceous cattle feed

The basidiomycete cultures, Polyporus strains BH₁ and BW₁, were cultivated on whole bagasse substrate in a solid-state system. The cultures degraded cellulose, hemicellulose and lignin contents of bagasse, increasing the digestibility and protein content of the product, to be utilized as cattle feed. Untreated and unwashed bagasse proved a good substrate.